NKT cell-activating agents containing α-glycosylceramides

ABSTRACT

An objective of the present invention is to provide NKT cell-activating agents, therapeutic agents for autoimmune diseases (for example, systemic lupus erythematosus, systemic sclerosis, ulcerative colitis, encephalomyelitis, multiple sclerosis and human type I diabetes), and abortifacients. The medicinal compositions according to the present invention comprise α-glycosylceramides of the following formula (I), or a salt or a solvate thereof as an active ingredient.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to NKT cell-activating agents, therapeuticagents for autoimmune diseases and agents for inducing abortion.

2. Background Art

It has been revealed that intermediate TCR cells (TCR^(int) cells),which express T-cell receptors (TCRs) intermediately, are related tonatural killer (NK) cells in terms of their features, for example,showing a large granular lymphocyte (LGL)-like morphology, constantlyexpressing IL-2R β-chains, and having perforin granules, but they areclearly different from NK cells in terms of having TCRs (Watanabe, H. etal., J. Immunol., 155, 2972 (1995)). Furthermore, among the TCR^(int)cells activated by interleukin 12 (IL-12), NK 1.1-expressing NK1.1⁺TCR^(int) (NKT) cells have been shown to be important effector cellsin controlling hematogenous metastases of tumors to the liver and lungin mice (Hashimoto, W. et al., J. Immunol., 154, 4333 (1995); Anzai, R.et al., Immunol., 88, 82 (1996)). These data suggest that the NKT cellsmay play an important role in eradicating cancer cells, parasites,protozoans, and intracellular infectious bacteria such as Listeriamonocytogenes and Micobacterium tubeculosis (Seki, S. et al., Clin.Immunol., 28, 1069 (1996)).

The NKT cells are also known to be closely associated with acuterejection in bone marrow transplantation (Yankelevich, B. et al., J.Immunol., 142, 3423 (1989)) and with controlling of IgE antibodyproduction by controlling Th1/Th2 differentiation of helper T cells(Yoshimoto, T. et al., J. Exp. Med., 179, 1285 (1994)). Thus, the NKTcells are a group of new cells that are currently attracting enormousattention.

Vα14⁺ NKT cells are a subset of the above-mentioned NKT cells. ManyVα14⁺ NKT cells express Vα14Jα281 mRNA and have this as a TCR α-chain.Recently, the Vα14⁺ NKT cells were shown to be closely associated withthe onset of autoimmune diseases. The number of Vα14⁺ NKT cells wasrevealed to selectively decrease prior to the onset of an autoimmunedisease in MRL 1pr/1pr mice, model mice for an autoimmune disease (humansystemic lupus erythematosus) in which abnormal lymphocytes accumulateat 17-20 weeks old (Mieza, M. A. et al., J. Immunol., 156, 4035 (1996)).

Similar phenomena were also observed in model mice for other autoimmunediseases, such as gld mice and (NZB×NZW) F1 mice, revealing that theVα14⁺ NKT cells are closely associated with the onset of autoimmunediseases (Makino, Y. et al., Clin. Immunol., 28, 1487 (1996)).

More interestingly, similar phenomena were also observed in humans. TheVα24JαQα chain, a human homologue to the mouse Vα14Jα281 chain, wasfound in peripheral blood CD4⁻/CD8⁻ T cells at a level of 20-50% inhealthy humans but not at all in sclerosis patients (Sumida, T. et al.,J. Exp. Med., 182, 1163 (1995)).

Thus, the mouse Vα14⁺ NKT cells and human Vα24JαQα T cells are known tobe involved in various autoimmune diseases which are caused by differentcausative genes or genetic background. Therefore, IL-12 having an NKTcell-activating activity as mentioned above was expected to be atherapeutic agent for autoimmune diseases such as human systemic lupuserythematosus (SLE) and systemic sclerosis (SSc). However, a markedincrease in the number of abnormal lymphocytes (CD3⁺B220⁺ doublenegative T cells) in the spleen and lymph nodes was observed in MRL1pr/1pr mice to which IL-12 was administered as compared with thecontrol mice (Takenori Tsutsui et al., Proceedings of Annual Meeting ofthe Japanese Society for Immunology, 347 (1996)).

β-Galactosylceramides or β-glucosylceramides, in which various sugarsbound to ceramides in a β-configuration, are present in the mammal body(Svennerholm, L. et al., Biochim. Biophys. Acta, 280, 626 (1972);Karlsson, K. -A. et al., Biochim. Biophys. Acta, 316, 317 (1973)). Onthe other hand, it is known that α-galactosylceramides have markedimmunostimulatory activity and antitumor activity (Morita, M. et al., J.Med. Chem., 38, 2176 (1995)) and such activities byα-galactosylceramides or α-glucosylceramides are known to be muchstronger than those by β-galactosylceramides or β-glucosylceramides(Motoki, K. et al., Biol. Pharm. Bull., 18, 1487 (1995)). It is alsoknown that administration of compounds having an α-glucosylceramidestructure protects the body from radiation (Motoki, K. et al., Bioorg.Med. Chem. Lett., 5, 2413 (1995)), suppresses the metastasis of mousemelanoma B16 to the lung (Kobayashi, E. et al., Oncology Res., 7, 529(1995)) and metastasis of mouse colon adenocarcinoma, Colon 26, andmouse T lymphoma EL-4 to the liver (Kazuhiro Motoki et al., Proceedingsof Annual Meeting of the Japanese Cancer Association, 523 (1996)), andincreases the number of platelets and leukocytes (Motoki, K. et al.,Biol. Pharm. Bull., 19, 952 (1996)).

However, there are no reports to date that compounds having anα-glucosylceramide structure are effective on autoimmune diseases, thatsuch compounds would induce abortion, or that such compounds could evenaffect NKT cells.

SUMMARY OF THE INVENTION

The present inventors have now found that α-glycosylceramides enhanceantitumor cytotoxic activity against tumor cells of NKT cells inRAG-1KO/Vα14tg/Vβ8.2tg mice (mice having a large number of NKT cells butneither B cells, T cells nor NK cells in the lymphocyte fraction),markedly increase the number of NKT cells, in particular, mouse Vα14⁺NKT cells and human Vα24⁺ NKT cells, suppress abnormal swelling ofaxillary and inguinal lymph nodes (accumulation of abnormal lymphocytes)in MRL 1pr/1pr mice which are considered model mice for human systemiclupus erythematosus, and control the progression of mouse colitisinduced with 4% DSS.

The present inventors have also found that α-glycosylceramides suppressthe onset of experimental autoimmune encephalomyelitis. Thisexperimental autoimmune encephalomyelitis in mice is a model for humanmultiple sclerosis. The present inventors have also found thatα-glycosylceramides suppress the spontaneous onset of diabetes in NODmice which are model animals for human type I diabetes.

The present inventors have further found that α-glycosylceramides havean aborting effect on pregnant mice.

An objective of the present invention is to provide an agent foractivating an NKT cell and an activated NKT cell.

Another objective of the present invention is to provide a therapeuticagent for autoimmune diseases such as systemic lupus erythematosus,systemic sclerosis, ulcerative colitis, encephalomyelitis, multiplesclerosis, or type I diabetes.

Further objective of the present invention is to provide an agent forinducing abortion.

The NKT cell-activating agent, the therapeutic agent for autoimmunediseases and the agent for inducing abortion according to the presentinvention comprise a compound of formula (I) or a salt or a solvatethereof:

wherein

R¹ represents H or OH,

X represents an integer between 7 and 27,

R² represents a substituent selected from the group consisting of thefollowing (a) to (e) (wherein Y represents an integer between 5 and 17):

(a) —CH₂(CH₂)_(Y)CH₃

(b) —CH(OH)(CH₂)_(Y)CH₃

(c) —CH(OH)(CH₂)_(Y)CH(CH₃)₂

(d) —CH═CH(CH₂)_(Y)CH₃

(e) —CH(OH)(CH₂)_(Y)CH(CH₃)CH₂CH₃, and

R³ to R⁹ represent substituents as defined in any one of the followingi) to v):

i) when R³, R⁶ and R⁸ represent H,

R⁴ represents H, OH, NH₂, NHCOCH₃, or a substituent selected from thegroup consisting of the following groups (A) to (D):

R⁵ represents OH or a substituent selected from the group consisting ofthe following groups (E) and (F):

R⁷ represents OH or a substituent selected from the group consisting ofthe following groups (A) to (D):

R⁹ represents H, CH₃, CH₂OH or a substituent selected from the groupconsisting of the following groups (A′) to (D′):

ii) when R³, R⁶ and R⁷ represent H,

R⁴ represents H, OH, NH₂, NHCOCH₃, or a substituent selected from thegroup consisting of the following groups (A) to (D):

R⁵ represents OH or a substituent selected from the group consisting ofgroups (E) and (F):

R⁸ represents OH or a substituent selected from the group consisting ofthe following groups (A) to (D):

R⁹represents H, CH₃, CH₂OH or a substituent selected from the groupconsisting of the following groups (A′) to (D′):

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows enhancing effect of KRN7000 on cytotoxic activity of NKTcells against tumor cells. The E/T ratio indicates the number ofeffector cells (spleen cell counts)/the number of target cells (YAC-1cell counts).

FIG. 2 shows stimulation of NKT cell proliferation by KRN 7000 in thespleen.

A: Results of FACS analysis of the spleen lymphocyte fraction. Thehorizontal axis indicates the fluorescence of FITC-labeled anti-TCRαβmonoclonal antibody and the vertical axis indicates the fluorescence ofcychrome-labeled anti-NK1.1 monoclonal antibody.

B: Results of FACS analysis of the spleen lymphocyte fraction. Thevertical axis indicates relative cell counts and the horizontal axisindicates the fluorescence of PE-labeled Vα14 monoclonal antibody. Thewhite area shows the fluorescence when stained with the PE-labeled Vα14monoclonal antibody after pretreating with an unlabeled Vα14 monoclonalantibody (cold blocking). The shaded area shows the fluorescencedistribution of the PE-labeled Vα14 monoclonal antibody to Vα14⁺ cellsafter the administration of vehicle or KRN 7000.

C: Change in the number of total cells, T cells, NK cells and Vα14⁺ NKTcells in the spleen lymphocyte fraction by the administration of KRN7000. :Vα14⁺ NKT cells, ∘:total cells, ⋄:T cells, □:NK cells.

FIG. 3 shows results as to the progression of lymphatic swelling-withtime in 1pr/1pr mice to which KRN 7000 was administered. Lymph nodes arescored into 4 grades, i.e., −(0), +(1), ++(2) and +++(3) depending onsize. Summed scores of the right and left sides of the axillary lymphnode (A) or inguinal lymph node (B) are shown as lymph node swellingindexes.

FIG. 4 shows survival rates of MRL lpr/lpr mice to which KRN 7000 wasadministered.

FIG. 5 shows the activity of KRN 7000 in suppressing colitis in miceinduced with 4% DSS. 4% DSS was continuously administered to the mice inthe drinking water during the experiment.

A: Change in the body weight of mice in different groups. B: Survivalrates of mice in different groups.

FIG. 6 shows the effect of KRN 7000 on experimental autoimmuneencephalomyelitis (EAE) induced in C57BL/6 mice, for example, by myelinoligodendroglia protein (MOG). A: Group to which vehicle wasadministered. B: Group to which KRN 7000 (20 μg/kg) was administered.EAE symptoms were scored as follows: Clinical scores: 0: normal, 1:paralysis in tails, 2: static reflex insufficiency, 3: paralysis in backfeet, 4: paralysis in front and back feet, 5: death.

FIG. 7 shows the effect of KRN 7000 on spontaneous diabetes in NOD mice.

FIG. 8 shows the stimulatory activity of KRN 7000 in the proliferationof Vα24⁺ NKT cells. After an autologous mixed lymphocyte reaction usingperipheral blood mononuclear cells as responding cells, CD4⁻CD8⁻ cellswere recovered to specify the phenotype using labeled antibodies. Dottedlines indicate the fluorescence distribution when stained with controlantibodies (mouse IgG or rat IgM) and solid lines indicate thefluorescence distribution when stained with anti-CD3, CD4, CD8, andVα24, Vβ11 antibodies (Immunotech), and anti-NKRP1A antibodies (BectonDickinson).

FIG. 9 shows stimulatory activity of KRN 7000 in Vα24⁺ NKT-cellproliferation. When antigen-presenting cells were treated with KRN 7000,stimulation of proliferation of Vα24⁺ NKT cells was observed in a mannerdependent on the number of the antigen-presenting cells.

FIG. 10 shows the outline of reactions for the synthesis of KRN 7000,the representative α-glycosylceramide compound used in the presentinvention. In the drawing, pyr represents pyridine, BrPPh₃ (CH₂)₁₂CH₃represents tridecanetriphenylphosphonium bromide, n-BuLi representsn-butyl lithium, MsCl represents methanesulfonyl chloride, BnBrrepresents benzyl bromide, and 1-PrOH represents propyl alcohol.

FIG. 11 is the continuation of the reactions for the synthesis as shownin FIG. 10. WSC—HCl represents1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide hydrochloride, MS4Arepresents molecular sieves 4A, and Hex4NBr representstetrahexylammonium bromide.

FIG. 12 shows chemical formulas of the compounds in Examples 1 to 3.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of Formula (I)

In the compounds of formula (I), X in the ceramide moiety preferablyrepresents an integer between 11 and 25.

Y in R² preferably represents an integer between 9 and 17, morepreferably between 11 and 15.

Preferable combinations for X and R² in the ceramide moiety of formula(I) are compounds in which X is an integer between 21 and 25 and R² issubstituent (b) (wherein Y is an integer between 11 and 15) andcompounds in which X is an integer between 9 and 13 and R² is thesubstituent (a) (wherein Y is an integer between 11 and 15).

Preferable combinations for R³ to R⁹ in the sugar moiety of formula (I)are compounds in which R³ and R⁶ are H, R⁴ is OH or any substituent ofgroups (A) to (D), R⁵ is OH or any substituent of group (E) or (F), R⁷and R⁸ are each H or OH (but R⁷ and R⁸ are different from one another),and R⁹ is CH₂OH, CH₃, H or any substituent of groups (A′) to (D′).

More preferable combinations include compounds in which R³ and R⁶ are H,R⁴ and R⁵ are OH, R⁷ and R⁸ are each H or OH (but R⁷ and R⁸ aredifferent from one another), and R⁹ is CH₂OH or any substituent ofgroups (A′) to (D′), and compounds in which R³, R⁶ and R⁸ are H, R⁴, R⁵and R⁷ are OH, and R⁹ is CH₂OH.

Preferable examples of compounds of formula (I) include compounds inwhich

X is an integer between 21 and 25,

R² is substituent (b) (wherein Y is an integer between 11 and 15),

R³ and R⁶ are H,

R⁴ is OH or a group selected from the group consisting of groups (A) to(D),

R⁵ is OH or a group selected from the group consisting of groups (E) and(F),

R⁷ and R⁸ are each H or OH (but both R⁷ and R⁸ are not the samesubstituent), and

R⁹ is CH₂OH or a group selected from the group consisting of groups (A′)to (D′);

compounds in which

X is an integer between 9 and 13,

R² is substituent (a) (wherein Y is an integer between 11 and 15),

R³ and R⁶ are H,

R⁴ and R⁵ are OH,

R⁷ and R9 are each H or OH (but both R⁷ and R⁸ are not the samesubstituent), and

R⁹ is H, CH₃ or CH₂OH;

compounds in which

X is an integer between 21 and 25,

R² is substituent (b) (wherein Y is an integer between 11 and 15),

R³ and R⁶ are H,

R⁴ and R⁵ are OH,

R⁷ and R⁸ are each H or OH (but both R⁷ and R⁸ are not the samesubstituent), and

R⁹ is CH₂OH or a group selected from the group consisting of groups (A′)to (D′); and

compounds in which

X is an integer between 21 and 25,

R² is substituent (b) (wherein Y is an integer between 11 and 15),

R³, R⁶ and R⁸ are H,

R⁴, R⁵ and R⁷ are OH, and

R⁹ is CH₂OH.

Preferable compounds as effective components of therapeutic agentsaccording to the present invention include

(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecanediol(KRN 7000),

(2S,3R)-1-(α-D-galactopyranosyloxy)-2-tetradecanoyl amino-3-octadecanol(AGL-517),

(2S,3R)-1-(α-D-glucopyranosyloxy)-2-tetradecanoyl amino-3-octadecanol(AGL-563),

(2S,3R)-1-(6′-deoxy-α-D-galactopyranosyloxy)-2-tetradecanoylamino-3-octadecanol (AGL-571),

(2S,3R)-1-(β-L-arabinopyranosyloxy)-2-tetradecanoyl amino-3-octadecanol(AGL-577),

O-α-D-galactopyranosyl-(1→6)-O-α-D-galactopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-hexacosanoyl-1,3,4-octadecanetriol (AGL-586),

O-α-D-galactopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-hexacosanoyl-1,3,4-octadecanetriol (AGL-584),

O-α-D-galactopyranosyl-(1→2)-O-α-D-galactopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-[(R)-2-hydroxytetracosanoyl]-1,3,4-octadecanetriol(S1140B-9),

O-β-D-galactofuranosyl-(1→3)-O-α-D-galactopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-[(R)-2-hydroxytetracosanoyl]-1,3,4-octadecanetriol (719-7), and

O-(N-acetyl-2-amino-2-deoxy-α-D-galactopyranosyl-(1→3)-O-[α-D-glucopyranosyl-(1→2)]-O-α-D-galactopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-[(R)-2-hydroxytetracosanoyl]-1,3,4-octadecanetriol (STL-8).

A particularly preferable compound used as an active ingredient intherapeutic agents according to the present invention is (2S, 3S,4R)-1-(α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecanediol(KRN 7000).

The compounds of formula (I) may be in the form of pharmaceuticallyacceptable nontoxic salts thereof. Salts of formula (I) include acidadded salts, such as salts with inorganic acids (e.g., hydrochloricacid, sulfuric acid, nitric acid and phosphoric acid) or with organicacids (e.g., acetic acid, propionic acid, maleic acid, oleic acid,palmitic acid, citric acid, succinic acid, tartaric acid, fumaric acid,glutamic acid, pantothenic acid, laurylsulfonic acid, methanesulfonicacid and phthalic acid).

The compounds of formula (I) may be in the form of solvates thereof(e.g., hydrates).

The compounds of formula (I) can be produced by any purposive method tosynthesize α-glycosylceramides.

First, a ceramide moiety is synthesized using D-lyxose as a startingmaterial, then a sugar is introduced into this ceramide to preparecompounds of formula (I). A general method to synthesize suchα-glycosylceramides can be found, for example, in WO93/5055, WO94/2168,WO/9020 and WO94/24142.

The compounds of formula (I) can also be isolated from natural products(e.g., biological organisms) and purified by column chromatography orthe like.

Use of Compounds of Formula (I)

The present inventors have found that antitumor cytotoxic activity ofNKT cells against tumor cells was enhanced when KRN 7000, arepresentative compound according to the present invention, wasadministered to RAG-1KO/Vα14tg/Vβ8.2tg mice (Pharmacological TestExample 1).

The present inventors have found that α-glycosylceramides markedlyincrease the number of NKT cells, particularly Vα14⁺ NKT cells andVα-24⁺ NKT cells (Pharmacological Test Examples 2, 6 and 9). Mouse Vα14⁺NKT cells and human Vα24⁺ NKT cells have been shown to be involved invarious autoimmune diseases caused by different causative genes andgenetic background as suggested by findings that Vα-14⁺ NKT cellsdecrease in autoimmune disease model mice, that Vα24⁺ JαQα T cellsdisappear in sclerosis patients, and that Vα24⁺ NKT cells greatlydecrease in patients with advanced type I diabetes (Mieza, M. A. et al.,J. Immunol., 156, 4035 (1996); Makino, Y. et al., Clin. Immunol., 28,1487 (1996); Sumida, T. et al., J. Exp. Med., 182, 1163 (1995); Wilsonet al., Nature, 391, 177 (1998)). The present inventors also found thatwhen KRN 7000 was administered to MRL lpr/lpr mice, which are consideredmodel mice for human systemic lupus erythematosus (Sakamoto, A. Clin.Immunol., 28, 1558 (1996)), abnormal swelling of axillary and inguinallymph nodes (accumulation of abnormal lymphocytes) was suppressed(Pharmacological Test Example 3). The abnormal swelling of lymph nodesis a characteristic symptom observed in MRL lpr/lpr mice with aging.

Accordingly, as the first aspect of the present invention, the compoundof formula (I), or a salt or a solvate thereof can be used as agents foractivating NKT cells. “NKT cells” as used herein include human Vα24⁺ NKTcells and mouse Vα14⁺ NKT cells. Human Vα24⁺ NKT cells are a subset ofhuman Vα24JαQα T cells and mean Vα24⁺ double negative (CD4⁻CD8⁻) T cells(Dellabona, P. et al., J.Exp. Med., 180, 1171 (1994)). Furthermore, theterm “NKT cell activation” or “activating NKT cell” includes enhancementof cytotoxic activity and stimulation of NKT cell proliferation.

As the second aspect of the present invention, the compound of formula(I), or a salt or a solvate thereof can be used as therapeutic agentsfor autoimmune diseases. The term “autoimmune diseases” as used hereininclude systemic lupus erythematosus, systemic sclerosis, ulcerativecolitis, multiple sclerosis, encephalomyelitis, type I diabetes, chronicarticular rheumatism, Sjoegren's syndrome, primary biliary cirrhosis,idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia,myasthenia gravis, sympathetic ophthalmia, Goodpasture's syndrome (e.g.,glomerular nephritis), pernicious anemia, and Hashimoto's disease. Theterm “treatment” or “therapy” as used herein includes “prevention”.

The compound of formula (I) and IL-12 induce abortion in pregnant mice(Pharmacological Test Example 10). Accordingly, as the third aspect ofthe present invention, the compound of formula (I) or a salt or asolvate thereof and IL-12 can be used as an agent for inducing abortion.The compound of formula (I) and IL-12 can be administered not only topregnant animals but also to animals that can get pregnant. Pregnancycan be suppressed by administering the compound of formula (I) or IL-12in advance to animals that can get pregnant. Accordingly, the term“agents for inducing abortion” means “contraceptives”.

The compound of formula (I) or a salt or a solvate thereof and IL-12 canbe formulated into suitable dosage forms depending on the medicaltreatment, administration route, and purpose of administration, e.g.,injectable agents, suspensions, emulsions, ointments, creams, tablets,capsules, granules, powders, pills, grains, troches, formulations forrectal administration, oily suppositories and water-solublesuppositories.

These various pharmaceutical formulations can be prepared byconventional methods using the following pharmaceutically acceptablevehicles or the like: excipients such as solvents (e.g., water,physiological saline), bulking agents and filling agents (e.g., lactose,starch, crystalline cellulose, mannitol, maltose, calciumhydrogenphosphate, soft silicic acid anhydride and calcium carbonate);auxiliaries such as solubilizing agents (e.g., ethanol andpolysolvates), binding agents (e.g., starch, polyvinyl pyrrolidine,hydroxypropyl cellulose, ethylcellulose, carboxymethyl cellulose and gumarabic), disintegrating agents (e.g., starch and carboxymethyl cellulosecalcium), lubricating agents (e.g., magnesium stearate, talc andhydrogenated oil), stabilizing agents (e.g., lactose, mannitol, maltose,polysolvates, macrogol, and polyoxyethylene hydrogenated castor oil),isotonic agents, wetting agents, lubricating agents, dispersing agents,buffering agents and solubilizing agents; and additives such asantioxidants, preservatives, flavoring and aromatizing agents, analgesicagents, stabilizing agents, coloring agents and sweetening agents.

If necessary, glycerol, dimethyacetamide, 70% sodium lactate,surfactants and alkaline substances (e.g., ethylenediamine, ethanolamine, sodium carbonate, arginine, meglumine and trisaminomethane) canalso be added to various pharmaceutical formulations.

In the present invention, the compound of formula (I) and IL-12 can beadministered via any purposive routes, for example, in the case ofanimals, intraperitoneal or subcutaneous administration, intravenous orintra-arterial administration and local administration by injection.Furthermore, in the case of humans, intravenous or intra-arterialadministration, local administration by injection, intraperitoneal orintrathoracic administration, subcutaneous administration, intramuscularadministration, sublingual administration, percutaneous absorption orrectal administration can be used. Intravenous administration is mostpreferable.

Individual effective components in therapeutic agents of the presentinvention can be administered continuously or intermittently dependingon individual situations. Actual doses are determined depending on avariety of factors such as the methods of administration, the conditionsof the patient, such as age, body weight, sex and sensitivity, time ofadministration, and other medicaments taken in combination. A daily doseof compounds of formula (I) and IL-12 for an adult human, for examplefor intravenous administration, is generally between about 0.001 and 10mg, preferably between 0.01 and 1 mg. The compound of formula (I) ispreferably formulated into freeze-dried preparations, which ispreferably dissolved with injection-grade distilled water immediatelybefore administration to patients.

The present invention provides methods of treating autoimmune diseases,which comprises the step of administering NKT cells activated by thecompound of formula (I), or a salt or a solvate thereof (activated NKTcells) to mammals, including humans.

Activated NKT cells can be obtained by culturing NKT cells in vitro inthe presence of the compound of formula (I), or a salt or a solvatethereof. Furthermore, activated NKT cells can be isolated from themammal body to which compounds of formula (I) are administered.

NKT cells, which are to be cultured in vitro with compounds of formula(I), can be isolated from healthy humans, patients or suspectedsufferers. For human treatment, NKT cells are preferably human Vα24⁺ NKTcells.

Activated NKT cells can be administered to mammals by implanting theactivated NKT cells in the animal's body, for example, the vein.

The present invention provides methods for activating NKT cells, whichcomprise the step of culturing NKT cells in vitro in the presence of acompound of formula (I), or a salt or a solvate thereof.

The present invention provides methods for inducing abortion, whichcomprise the step of administering the compound of formula (I) or a saltor a solvate thereof, or IL-12 to mammals, including humans.

EXAMPLES

The present invention is further illustrated by the following examplesthat are not intended as a limitation of the invention.

Synthesis, Isolation and Purification of Compounds

Example 1 Synthesis of (2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecanediol (KRN 7000)

The synthesizing steps are shown in FIGS. 10 and 11.

(1) Synthesis of Compound G1

Sulfuric acid (0.5 ml) was added to a solution of D-lyxose (200 g, 1.33mol) in acetone (3.0 L), which had been dried with calcium chloride, andthe admixture was stirred for 18 hours at a room temperature. Molecularsieves 4A powder (100 g) was added, the reaction mixture wasneutralized, then filtered with Celite, and the resulting residue waswashed with acetone. The filtrate and the wash were combined andconcentrated under vacuum to obtain a crude product of G1. Yield 240 g(95%). The product was used for the next step without furtherpurification. A sample for assay was purified by silica gelchromatography using hexane:acetone (9:1) as the eluting solvent.

mp76-78° C.; FDMS m/z 191(M+1)⁺; ¹H-NMR(500 MHz,CDCl₃) δ5.45(1H,d,J=1.8Hz),4.83(1H,dd,J=3.7,5.5 Hz),4.64(1H,d,J=6.1Hz),4.27-4.30(1H,m),3.90-3.99(2H,m),1.48(3H,s), 1.32(3H,s)

(2) Synthesis of Compound G2

Pyridine (10 ml) and trityl chloride (39.0 g) were added to a methylenechloride solution (168 ml) of compound G1 (239 g, about 1.26 mmol), andthe admixture was stirred for 4 hours at 32° C. Ethanol (8 ml) was addeddropwise, and the admixture was stirred for 2 hours at a roomtemperature. After washing with an aqueous saturated ammonium chloridesolution, an aqueous saturated sodium hydrogencarbonate solution and asaline solution, concentration under vacuum was carried out. Theresulting residue was dissolved in ethyl acetate, cooled to 0° C. andthen crystallized. Yield 501 g (87% from D-lyxose).

mp174-176° C.;FDMS m/z 432M⁺; ¹H-NMR(500 MHz,CDCl₃)δ7.21-7.49(15H,m),5.38(1H,d,J=2.4 Hz), 4.75(1H,dd,J=3.7,6.1 Hz), 4.59(1H,d,J=6.1Hz),4.31-4.35(1H,m), 3.43(1H,dd,J=4.9, 9.8 Hz),3.39(1H,dd,J=6.7,9.8 Hz),1.29(3H,s), 1.28(3H,s)

(3) Synthesis of Compound G3

To a THF solution (1500 ml) of tridecanetriphenylphosphonium bromide(962 g, 1.16 mol; prepared by heating 1-bromotridecane andtriphenylphosphine for 4.5 hours at 140° C.), a 2.5 M hexane solution ofn-butyl lithium (462 ml, 366 mmol) was added dropwise at 0° C. under anargon atmosphere. The admixture was stirred for 15 minutes, then a THFsolution (450 ml) of compound G2 (250 g, 579 mmol) was added dropwise.This admixture was stirred for 18 hours while gradually raising thetemperature to room temperature. The reaction solution was concentratedunder vacuum, a mixture of hexane:methanol:water (10:7:3, 1000 ml) wasadded to the residue, and the admixture was washed with an aqueoussaturated ammonium chloride solution. The water layer was extracted withhexane (500 ml). All the organic layers were combined, dried overanhydrous magnesium sulfate, and then concentrated under vacuum toobtain a crude product of compound G3. The product was used for the nextstep without further purification. Yield 339 g (98%). A sample for assaywas purified by silica gel chromatography using hexane:ethyl acetate(9:1) as the eluting solvent.

FDMS m/z 598M⁺; ¹H-NMR(500 MHz,CDCl₃)δ7.21-7.45(15H,m),5.48-5.59(2H,m),4.91(0.7H,t,J=7.3 Hz),4.44(0.3H,t,J=7.3 Hz),4.26(0.3H,dd,J=4.3,7.3 Hz),4.21(0.7H,dd,J=4.3,6.7 Hz),3.75(0.7H,m),3.69(0.3H,m),3.24(0.3H,dd,J=4.9,9.8Hz),3.17(0.7H,dd,J=4.9,9.8 Hz),3.09-3.14[1H,(3.11,dd,J=4.9,9.2 Hz),H1bEoverlapped],1.75-2.03(2H,m),1.49(3H,s),1.39 and 1.38 (3H,eachs),1.21-1.34 (20H,m),0.88(3H,t,J=6.7 Hz)

(4) Synthesis of Compound G4

To a methylene chloride solution (1500 ml) of compound G3 (338 g, about565 mol), pyridine (500 ml) was added, and methanesulfonyl chloride (49ml, 633 mmol) was added dropwise. The admixture was stirred for 24 hoursat 31° C. Ethanol (40 ml) was added dropwise and the admixture wasstirred for 1 hour at a room temperature. After concentration undervacuum, a mixture of hexane:methanol:water (10:7:3, 1000 ml) was addedto the residue for separation. The water layer was extracted 3 timeswith hexane (200 ml). All the organic layers were combined, dried overanhydrous magnesium sulfate, and then concentrated under vacuum toobtain a crude product of compound G4. The product was used for the nextstep without further purification. Yield 363 g (95%). A sample for assaywas purified by silica gel chromatography using hexane:ethyl acetate(9:1) as the eluting solvent.

FDMS m/z 676M⁺; ¹H-NMR(500 MHz,CDCl₃)δ7.21-7.47(15H,m),5.41(0.7H,ddd,J=5.5,9.2,11.0 Hz),5.32(0.7H,bt,J=11.0Hz),5.22(0.3H,bdd,J=9.2,15.0 Hz),5.02(0.3H,dt,Jt=7.3 Hz,Jd=15.0 Hz),4.8(0.7H,ddd,J=3.1,5.5,7.9 Hz),4.73(0.7H,dd,J=5.5,9.8 Hz),4.64-4.67(0.3H,m),4.61(0.3H,dd,J=5.5,9.2 Hz), 4.48(0.7H,dd,J=5.5,7.9Hz),4.22(0.3H,dd,J=5.5,9.2 Hz), 3.55(0.3H,dd,J=2.4,11.6Hz),3.45(0.7H,dd,J=3.2,11.0 Hz),3.06-3.12[4H,(3.12,s),(3.11,s),(3.09,dd,J=3.1,11.0 Hz)],1.66-1.82(2H,m),1.47 and 1.46(3H,each s),1.39(3H,s),1.13-1.35(20H,m),0.88(3H,t,J=6.8 Hz)

(5) Synthesis of Compound G5

To a methylene chloride solution (1500 ml) of compound G4 (362 g, about536 mol), methanol (350 ml) was added, then concentrated hydrochloricacid (200 ml) was added dropwise. The admixture was stirred for 5 hoursat a room temperature. The reaction solution was neutralized by addingsodium hydrogencarbonate, then filtered. The filtrate was concentratedunder vacuum and ethyl acetate was added to the resulting residue andwashing was carried out with a saline solution. The water layer wasextracted with ethyl acetate, all the organic layers were combined,dried over anhydrous magnesium sulfate, then concentrated under vacuum.Crystallization was carried out with hexane. Yield 161 g (70% from G2).

mp66-67° C.;FDMS m/z 377(M-H₂O)⁺; ¹H-NMR(500 MHz,CDCl₃+D₂O)δ5.86(0.3H,dt,Jt=7.3 Hz,Jd=14.7 Hz),5.77(0.7H,dt,Jt=7.3,Jd=10.4Hz),5.55(0.3H,br.dd,J=7.3,14.7 Hz),5.49(0.7H,bt,J=9.8Hz),4.91-4.97(1H,m),4.51(0.7H,bt,J=9.8 Hz),4.11(0.3H,bt, J=7.3Hz),3.94-4.03(2H,m),3.67-3.73[1H,(3.70,dd,J=3.1, 6.7Hz),(3.69,dd,J=3.1,7.3 Hz)],3.20 and 3.19(3H, eachs),2.05-2.22(2H,m),1.22-1.43(20H,m),0.88(3H,t,J=6.7 Hz)

(6) Synthesis of Compound G6

To a THF solution (780 ml) of compound G5 (160 g, about 405 mol), 5%palladium-barium sulfate (16 g) was added. After replacing the air in areaction chamber with hydrogen gas, the admixture was stirred for 20hours at a room temperature. The reaction solution was filtered usingCelite, then washed with a mixture of chloroform:methanol (1:1). Thefiltrate and wash were combined and concentrated under vacuum. Theresulting residue was crystallized with ethyl acetate. Yield 146 g(91%).

[α]²³ _(D)+12° (cl,CHCl₃/MeOH=1:1);mpl24-126° C;FDMS m/z397(M+1)⁺;¹H-NMR(500 MHz,CDCl₃/CD₃OD=1:1)δ4.93-4.96(1H,m,H2),3.91(1H,dd,J=6.7,12.2 Hz),3.85(1H,dd,J=4.9,12.2Hz),3.54-3.60(1H,m),3.50 (IH,dd,J=1.8,8.5 Hz), 3.19(3H,s),1.75-1.83(1H,m),1.53-1.62(1H,m),1.21-1.45(24H,m),0.89 (3H,t,J=6.7Hz)

(7) Synthesis of Compound G7

To a DMF solution (1000 ml) of compound G6 (145 g, 365 mol), sodiumazide (47 g, 730 mmol) was added, and the admixture was stirred for 4hours at 95° C. The reaction solution was concentrated, ethyl acetatewas added to the resulting residue and washing was carried out withwater. The water layer was extracted again with ethyl acetate. All theorganic layers were combined, washed with a saline solution, dried overanhydrous magnesium sulfate, and then concentrated under vacuum toobtain a crude product of compound G7. Yield 122 g (97%). The productwas used for the next step without further purification. Yield 126 g(95%). A sample for assay was purified by silica gel chromatographyusing hexane: ethyl acetate (9:1) as the eluting solvent.

[α]²³ _(D)+16.5° (c0.5,CHCl₃-MeOH,1:1);mp92-93° C.;FDMS m/z344(M+1)⁺;¹H-NMR(500 MHz,CD₃OD)δ3.91(1H,dd,J=3.7,11.6 Hz), 3.75(1H,dd,J=7.9,11.6 Hz), 3.49-3.61(3H,m), 1.50-1.71(2H,m),1.22-1.46(24H,m), 0.90(3H,t,J=6.7 Hz)

(8) Synthesis of Compound G8

To a methylene chloride solution (750 ml) of compound G7 (121 g, about352 mmol), pyridine (250 ml) and trityl chloride (124 g, 445 mmol) wereadded, and the admixture was stirred for 16 hours at a room temperature.Ethanol (30 ml) was added dropwise. The admixture was stirred for 30minutes at a room temperature, washed with an aqueous saturated sodiumhydrogencarbonate solution, an aqueous saturated ammonium chloridesolution and a saline solution, dried over anhydrous magnesium sulfate,and then concentrated under vacuum. The residue was purified by silicagel chromatography using hexane:ethyl acetate (10:1) as the elutingsolvent. Yield 34.4 g (52% from G6).

[α]²⁴ _(D)+11.9° (c0.9,CHCl₃),FDMS m/z 585M⁺; ¹H-NMR(500MHz,CDCl₃+D₂O)δ7.24-7.61(15H,m),3.62-3.66(2H,m),3.51-3.57(2H,m),3.42(1H,dd,J=6.0,10.4 Hz),1.23-1.56(26H,m),0.88(3H,t,J=6.7 Hz)

(9) Synthesis of Compound G9

To a DMF solution (300 ml) of compound G8 (33.5 g, 57.3 mmol), 60%hydrogenated sodium (5.5 g, about 138 mmol as NaH) was added, and theadmixture was stirred for 40 minutes at a room temperature. The reactionsolution was cooled to 0° C. and benzyl chloride (15 ml, 120 mmol) wasadded dropwise. The admixture was stirred for 18 hours while graduallyraising the temperature to a room temperature. Ice water (100 ml) wasadded to the reaction solution. After the reaction was stopped,extraction was carried out using ethyl acetate. The extract was washed 3times with a saline solution, and all the organic layers were combined,dried over anhydrous magnesium sulfate, and then concentrated undervacuum to obtain a crude product of compound G9. The product was usedfor the next step without further purification. Yield 42.2 g (96%). Asample for assay was purified by silica gel chromatography usinghexane:ethyl acetate (100:1) as the eluting solvent.

[α]²⁴ _(D)+9.8° (c1.0,CHCl₃),FDMS m/z 738(M-N₂)⁺; ¹H-NMR(500 MHz,CDCl₃)δ7.07-7.48(25H,m),4.57(1H,d,J=11.6 Hz),4.44(1H,d, J=11.6Hz),4.41(2H,s),3.73-3.79(1H,m),3.46-3.56(2H,m),3.37 (1H,dd,J=8.6,10.4Hz),1.20-1.64(26H,m),0.88(3H,t,J=6.7 Hz)

(10) Synthesis of Compounds G10 and G11

To a 1-propanol solution (250 ml) of compound G9 (41.2 g, about 54mmol), methanol (30 ml) was added, and further 5% palladium carbon (4.1g) and ammonium formate (27.1 g, 4.3 mol) were added. After stirring for16 hours at a room temperature, the admixture was diluted with ethylacetate and filtered with Celite. The filtrate was concentrated undervacuum, and the resulting residue was dissolved with ethyl acetate andwashed 3 times with an aqueous saturated sodium hydrogencarbonatesolution and a saline solution. All the organic layers were combined,dried over anhydrous magnesium sulfate, and then concentrated undervacuum to obtain a crude product of G10. Yield 38.9 g (98%). G10 thusobtained was used for the next step without further purification.

To a methylene chloride solution (300 ml) of compound G10, hexacosanoicacid (22.4 g, 56.5 mmol) and WSC hydrogenchloride (12.6 g, 64.6 mmol)were added, and the admixture was fluxed for 2 hours while heating. Themixture was cooled to room temperature and concentrated under vacuum.Ethyl acetate (500 ml) was added to the residue, and washing was carriedout with an aqueous 0.5 M hydrochloric acid solution, a saline solution,and an aqueous saturated sodium hydrogencarbonate solution, and furtherwith a saline solution. All the organic layers were combined, dried overanhydrous magnesium sulfate, and then concentrated under vacuum toobtain a crude product of compound G11. Yield 53.2 g (88%). G11 thusobtained was used for the next step without further purification. Asample for assay was purified by silica gel chromatography usinghexane:ethyl acetate (100:1) as the eluting solvent.

[α]²⁴ _(D)+5.3° (c0.4,CHCl₃);FDMS m/z 1118M⁺; ¹H-NMR(500 MHz,CDCl₃)δ7.20-7.38(25H,m),5.57(1H,d,J=9.1 Hz),4.80(1H,d, J=11.6Hz),4.48-4.50(3H,m),4.24-4.32(1H,m),3.83(1H,dd, J=3.0,6.7Hz),3.43-3.51(2H,m,H1a), 3.29(1H,dd,J=4.3,9.8 Hz), 1.92(2H,t,J=7.3 Hz),1.28-1.60(72H,m), 0.88(6H,t,J=6.7 Hz)

(11) Synthesis of Compound G12

To a methylene chloride solution (180 ml) of compound G11 (52.2 g, about47 mmol), methanol (36 ml) was added, then a 10% methanol chloridesolution (3.0 ml) was added dropwise, and the admixture was stirred for2 hours at a room temperature. The reaction solution was neutralizedwith sodium hydrogencarbonate powder (18 g) and filtered with Celite.The residue was washed with methylene chloride. The filtrate and washwere combined and washed with a saline solution. The organic layer wasdried over anhydrous magnesium sulfate, and then concentrated undervacuum. The residue was dissolved in acetone while heating, and thesolution was cooled to 0° C. and purified by precipitation. Yield 38.6 g(77% from G9).

[α]²⁴ _(D)−29.7° (c0.7,CHCl₃);mp75-76.5° C.;FDMS m/z 876M⁺; ¹H-NMR (500MHz,CDCl₃)δ7.30-0.47(10H,m),6.03(1H,d,J=7.9 Hz),4.72(1H,d,J=11.6Hz),4.66(1H,d,J=11.6 Hz),4.61(1H,d,J=11.6 Hz),4.45 (1H,d,J=11.6Hz),4.12-4.17(1H,m),4.00(1H,dt,Jt=4.3, Jd=7.3Hz),3.67-3.72(2H,m),3.61(1H,ddd,J=4.3,8.6,11.6 Hz),1.94-2.05(2H,m),1.15-1.69 (72H,m),0.88(6H,t,J=6.1 Hz)

(12) Synthesis of Compound G13

1) 2,3,4,6-tetra-O-benzyl-D-galactopyranosylacetate (79.8 g) wasdissolved in a mixture of toluene (160 ml) and isopropyl ether (520 ml),and the solution was cooled to −10 to 0° C. To this solution, anisopropyl ether solution (2.8 mmol/ml, about 100 ml) containing 2.0equivalent volumes of HBr was added. After stirring for about 90 minutesat −10 to 0° C., an aqueous 5% sodium hydrogencarbonate solution waspoured into the reaction solution, and excessive HBr was neutralized bystirring. The whole volume was transferred into a separation funnel forseparation, then the water layer was discarded and washing was carried 2times with an aqueous 10% sodium chloride solution. After concentrationunder vacuum, 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl bromide(GalBr) was obtained as a syrup.

2) DMF (140 ml), then a toluene solution (250 ml) of GalBr (about 137mmol) were added to a toluene solution (420 ml) of compound G12 (60.0 g,68.6 mmol), tetrahexylammonium bromide (89.4 g, 206 mmol) and molecularsieves 4A (60 g). The admixture was stirred for 72 hours at a roomtemperature. Methanol (12 ml) was added to the reaction solution, andthe admixture was stirred for 2 hours. Filtration with Celite andwashing with an aqueous saturated sodium hydrogencarbonate solution anda saline solution were followed by drying on anhydrous magnesium sulfateand concentration under vacuum. Acetonitrile was added to the resultingresidue and the admixture was stirred for 2 hours. The resultingprecipitate was dried under vacuum to obtain a dry powder. This powderwas purified by silica gel chromatography using hexane:ethyl acetate(8:1) as the eluting solvent. Yield 70.9 g (74%).

[α]²⁴ _(D)+18.8° (c0.9,CHCl₃);mp74-75° C.;FDMS m/z 1399(M+1)⁺;¹H-NMR(500 MHz,CDCl₃)δ7.21-7.37(30H,m),6.12(1H,d,J=9.0Hz),4.91(1H,d,J=11.6 Hz),4.84(1H,d,J=3.7Hz),4.72-4.80(4H,m),4.35-4.65(7H,m),4.12-4.18(1H,m),3.99-4.05(2H,m),3.84-3.93(4H,m),3.73(1H,dd,J=3.7, 11.0Hz),3.47-3.51(2H,m),3.42(1H,dd,J=6.1,9.1 Hz),1.87-1.99(2H,m),1.18-1.70(72H,m),0.88(6H,t,J=7.4 Hz)

(13) Synthesis of Compound KRN 7000

Compound G13 (60.0 g, 42.9 mmol) was added to ethanol (960 ml) to make asuspension, to which an ethanol suspension of 20% hydroxy palladium (6.0g) was added. Further, a hydrogen source, 4-methylcyclohexene (120 ml,93.5 mmol) was added. After fluxing for 4 hours while heating,filtration was carried out, and the solvent was removed. The residue waswashed with heated ethanol. The filtrate was allowed to stand at a roomtemperature to obtain a white precipitate, and the precipitate wasfiltered and dried under vacuum. The resulting powder was suspended inethanol:water (92:8, 3.5 L) and dissolved by heat while stirring. Thesolution was allowed to stand to obtain a precipitate again. Thesolution with the precipitate was filtered, and the filtrated cake wasdried under vacuum to obtain a white powder. Yield 35.0 g (95%).

[α]²³ _(D)+43.6° (c1.0,pyridine);mp189.5-190.5° C.; negative FABMS m/z857(M-H)⁻;IR(cm⁻¹,KBr)3300,2930,2850,1640,1540, 1470,1070;¹H-NMR(500MHz,C₅D₅N)δ8.47(1H,d,J=8.5 Hz), 5.58(1H,d,J=3.7Hz),5.27(1H,m),4.63-4.70(2H,m),4.56(1H,m), 4.52(1H,t,J=6.1Hz),4.37-4.47(4H,m),4.33(2H,m),2.45(2H,t, J=7.3Hz),2.25-2.34(1H,m),1.87-1.97(2H,m),1.78-1.85(2H,m),1.62-1.72(1H,m),1.26-1.45(66H,m), 0.88(6H,t,J=6.7 Hz),¹³C-NMR(125MHz,C₅D₅N)δ173.2(s),101.5(d),76.7(d),73.0(d),72.5(d),71.6(d),71.0(d),70.3(d),68.7(t),62.7(t),51.4(d),36.8(t),34.4(t),32.1(t),30.4(t),30.2(t),30.03(t),30.00(t),29.93(t),29.87(t),29.81(t),29.76(t),29.6(t),26.5(t),26.4(t),22.9(t),14.3(q)

Example 2 Isolation and Purification ofO-α-D-galactopyranosyl-(1→2)-O-α-D-galactopyranosyl-(1→1)-(2S,3S,4R)-2-amino-N-[(R)-2-hydroxytetracosanoyl]-1,3,4-octadecanetriol(S1140B-9)

A freeze dried powder (447.1 g) of sponges, which were harvested at adepth of 15-25 m from the sea near Kume Island of Okinawa Prefecture,was extracted with a mixture of chloroform and methanol, then theextracted liquid was concentrated under vacuum to obtain 51.28 g ofextract. The extract was partitioned with ethyl acetate and water, andthe upper layer and the middle layer were dried over anhydrous sodiumsulfate and concentrated under vacuum to obtain 18.37 g and 9.44 g offractions, respectively. An alcohol layer, which was obtained bypartitioning the fraction obtained from the upper layer with 10% aqueousmethanol and n-hexane, and the fraction obtained from the middle layerwere combined and concentrated. By repeating silica gel chromatography,169.9 mg of a single active component on normal phase TLC was obtained.Further purification was carried out by reversed phase HPLC using anODS-AM column (a product of YMC, 250 mm×20 mm diameter, methanol, 9.0ml/min) (retention time: 30.3 minutes) to obtain 10.2 mg of the purifiedtitle compound (S1140B-9).

The title compound can also be isolated and purified by the methoddescribed in F. Cafieri et al., Liebigs Ann. Chem. 1995, 1477-1481.

negative FABMS m/z 1007[(M-H)⁻];IR;¹HNMR(500 MHz,C₅D₅N,24° C.) δ(ppm)8.55(1H,d,J=9.2 Hz,NH),5.60(1H,d,J=3.7 Hz,H1″),5.57(1H,d,J=3.7Hz,H1′″),5.13(1H,m,H2),4.75(1H,dd,J=3.7,10.4 Hz,H2″),4.62(2H,m),4.54(4H,m),4.25-4.47(10H,m),2.17(2H,m),1.99(1H,m),1.87(2H,m),1.75(1H,m),1.65(2H,m),1.12-1.49(60H,m),0.85(6H,m,terminalmethyl);¹³C NMR(125 MHz,C₅D₅N,45° C.)δ(ppm)175.5(s,C1′),99.5(d,C1′″),98.6(d,C1″),76.7(d,C2″),76.0(d,C3),72.8(d,C4),72.6(d,C5″),72.6(d,C4″),72.5(d,C2),71.3(d,C3′″),71.0(d),70.8(d),70.5(d,C2′″),69.7(d,C3″),68.6(t,C1),62.7(t),62.5(t),51.2(t,C2),39.4(t),35.6(t),33.7(t),32.2(t),30.5(t),30.3(t),30.1(t),30.0(t),29.7(t),29.6(t),26.7(t),26.0(t),23.0(t),22.9(t),14.3(q,terminalmethyl)

Example 3

The following compounds were synthesized according to the methodsdescribed in the references given on the right column.

Compound name Reference (2S, 3R)-1-(α-D-galactopyranosyloxy)-2-W093/5055 tetradecanoylamino-3-octadecanol (AGL-517) (2S,3R)-1-(α-D-glucopyranosyloxy)-2- W094/9020tetradecanoylamino-3-octadecanol (AGL-563) (2S,3R)-1-(6′-deoxy-α-D-galactopyranosyloxy)- W094/90202-tetradecanoylamino-3-octadecanol (AGL-571) (2S,3R)-1-(β-L-arabinopyranosyloxy)-2- W094/9020tetradecanoylamino-3-octadecanol (AGL-577)O-α-D-galactopyranosyl-(1→6)-O-α-D- W094/24142galactopyranosyl-(1→1)-(2S, 3S, 4R)-2-amino-N-hexacosanoyl-1,3,4-octadecanetriol (AGL-586)O-α-D-galactopyranosyl-(1→6)-O-α-D- W094/24142 glucopyranosyl-(1→1)-(2S,3S, 4R)-2-amino-N- hexacosanoyl-1,3,4-octadecanetriol (AGL-584)O-α-D-galactofuranosyl-(1→3)-O-α-D- W094/24142galactopyranosyl-(1→3)-O-α-D-galactopyranosyl- (1→1)-(2S, 3S,4R)-2-amino-N-[(R)-2- hydroxytetracosanoyl]-1,3,4-octadecanetriol(719-7) O-(N-acetyl-2-amino-2-deoxy-α-D- W094/24142galactopyronosyl-(1→3)-O-[α-D-glucopyranosyl-(1→2)]-O-α-D-galactopyranosyl-(1→1)-(2S, 3S,4R)-2-amino-N-[(R)-2-hydroxytetracosanoyl]- 1,3,4-octadecanetriol(STL-8)

Relations between compounds of formula (I) and the compounds describedin the example mentioned above are shown in Table 1.

TABLE 1 X R1 R2 R3 R4 R5 R6 R7 R8 R9 KRN7000 23 H (b)Y = 13 H OH OH H OHH CH₂OH AGL517 11 H (a)Y = 13 H OH OH H OH H CH₂OH AGL563 11 H (a)Y = 13H OH OH H H OH CH₂OH AGL571 11 H (a)Y = 13 H OH OH H OH H CH₃ AGL577 11H (a)Y = 13 H OH OH H OH H H AGL586 23 H (b)Y = 13 H OH OH H OH HGroup(A′) AGL584 23 H (b)Y = 13 H OH OH H H OH Group(A′) S1140B-9 21 OH(b)Y = 13 H Group(A) OH H OH H CH₂OH 719-7 21 OH (b)Y = 13 H OH Group(E)H OH H CH₂OH STL-8 23 OH (b)Y = 13 H Group(B) Group(F) H OH H CH₂OH

Biological Test

Pharmacological Test Example 1 Enhancing Effect of KRN7000 on CytotoxicActivity of NKT Cells Against Tumor Cells

The following experiment was carried out using the compound of Example 1(KRN 7000) as a representative glycosidic compound of the presentinvention.

A vehicle (physiological saline containing 0.025% Polysolvate 20) or 100μg/kg of KRN 7000 was intravenously administered toRAG-1KO/Vα14tg/Vβ8.2tg mice (bearing a large number of NKT cells but notB cells, T cells or NK cells in their lymphocyte fraction), and 24 hourslater, the spleen was taken out from each mouse to prepare spleen cellsby conventional methods. RAG-1KO/Vα14tg/Vβ8.2tg mice were established bydeletion of the RAG-1 gene and forcible expression of Vα14 and Vβ8.2gene (Kawano T. et al., Science, 278, 1626-1629 (1997)). These mice areavailable from Masaru Taniguchi, School of Medicine, Chiba University.Cytotoxic activity of these spleen cells to mouse lymphoma YAC-1 cellswas studied by the 4 hour-⁵¹Cr-release method (Kobayashi, E. et al.,Oncology Res., 7, 529 (1955)). Results are shown in FIG. 1.

As shown in FIG. 1, cytotoxic activity against YAC-1 cells wassignificantly higher in the spleen cells prepared from the mice to whichKRN 7000 was administered than in those prepared from the mice to whichthe vehicle was administered.

These results indicate that KRN 7000 enhances cytotoxic activity ofspleen NKT cells against tumor cells, considering thatRAG-1KO/Vα14tg/β8.2tg mice bear a large number of NKT cells but no Bcells, T cells or NK cells in the lymphocyte fraction of the spleencells.

The results mentioned above indicate that KRN 7000 has an ability toenhance lytic activity of NKT cells against tumor cells.

Pharmacological Test Example 2 Stimulation of Spleen NKT CellProliferation by KRN 7000

A vehicle (physiological saline solution containing 0.5% Polysolvate 20)or 1, 10 or 100 ng/kg of KRN 7000 was intravenously administered toC57BL/6 mice (Japan SLC, Inc.), and the spleen of individual mice wastaken out after 24 hours to prepare spleen cells by conventionalmethods. These spleen cells were incubated in a plastic dish for 30minutes to prepare nonadherent cells. By removing B cells in thisnonadherent cells, a lymphocyte fraction was prepared. T cells, NKcells, NKT cells and Vα14⁺ NKT cells in this fraction were analyzed bythe 3-color FACS analysis using the FITC-labeled anti-TCR αβ monoclonalantibody (Pharmingen), cychrome-labeled anti-NK1.1 monoclonal antibody(Pharmingen) and PE-labeled anti-Vα14 monoclonal antibody (FIG. 2). Theanti-Vα14 monoclonal antibody was obtained by implanting anti-Vα14monoclonal antibody producing hybridomas (CMS-1; available from MasaruTaniguchi et al, School of Medicine, Chiba University) in nude mice andrecovering ascites from the animals to purify the antibody. In FIGS. 2Aand 2B, the numbers of NK1.1 cells, TCRαβ cells and Vα14 cells in thelymphocyte fraction were expressed as fluorescence intensity of thelabeled antibodies against these cells.

As shown in FIG. 2A, a marked increase in the rate of NK1.1⁺ TCRαβ⁺cells in the spleen lymphocyte fraction was observed in animals to which100 ng/kg of KRN 7000 were administered as compared with animals towhich the vehicle was administered.

As shown in FIG. 2B, an increase in the rate of Vα14⁺ cells in NK1.1⁺TCRαβ⁺ cells was clearly observed in animals to which 100 ng/kg of KRN7000 was administered as compared with animals to which the vehicle wasadministered.

Furthermore, as shown in FIG. 2C, the numbers of NK cells in the spleenlymphocyte fractions from the mice to which 10 or 100 ng/kg of KRN 7000is administered is equal to those from the vehicle-administered mice.However, an about 2-fold increase in the numbers of lymphocyte fractionsand T cells was observed in the KRN 7000-administered mice than thevehicle-administered mice. Further, the numbers of Vα14⁻ NKT cells andVα14⁺ NKT cells in the spleen lymphocyte fraction from the KRN7000-administered mice increased about more than three times (data notshown) and more than four times, respectively, as compared with thosefrom the vehicle-administered mice.

Further, analyses of T cells, NK cells, Vα14⁻ NKT cells and Vα14⁺ NKTcells in the liver showed a marked increase in the numbers of Vα14⁻ NKTcells and Vα14⁺ cells by KRN7000 administration, similarly to the caseof the spleen (data not shown).

The results mentioned above indicate that KRN 7000 has an ability toincrease the number of NKT cells, particularly Vα14⁺ NKT cells, in thebody.

Pharmacological Test Example 3 Suppression of Lymph Node Swelling in MRLlpr/lpr Mice by KRN 7000

Ten animals per group of female MRL lpr/lpr mice (Sakamoto, A. Clin.Immunol., 28, 1558 (1966)), were used for the following experiment.During the observation of 21 MRL mice purchased at the age of 6 weeks,swelling of the axillary lymph nodes was recognized in one mouse at theage of 10 weeks. Accordingly, the other 20 mice were randomly dividedinto 2 groups. A vehicle (a physiological saline solution containing0.025% Polysolvate 20) or KRN 7000 (100 μg/kg) was intraperitoneallyadministered twice a week (on Tuesday and Friday) to the abovementioned2 groups of animals starting from the age of 11 weeks. Axillary andinguinal lymph nodes were examined twice a week to observe the progressof lymph node swelling with time. The lymph nodes were scored into 4grades, i.e., −(0), +(1), ++(2) and +++(3) as a function of size. Thetotal scores of both right and left sides of the axillary lymph node (A)or inguinal lymph node (B) were expressed as lymph node swelling indexesas shown in FIG. 3.

As shown in FIG. 3A, the swelling in the axillary lymph nodes in MRLmice with aging was clearly suppressed by the administration of KRN7000. Further, as shown in FIG. 3B, the swelling in the inguinal lymphnodes in MRL mice with aging was also clearly suppressed by theadministration of KRN 7000. In other words, KRN 7000 clearly has acapability to suppress lymph node swelling in MRL mice.

The MRL mouse is a model mouse for human systemic lupus erythematosus(Sakamoto, A., Clin. Immun., 28, 1558 (1996)). The results mentionedabove indicate that KRN 7000 is effective in treating systemic lupuserythematosus.

Pharmacological Test Example 4 Effect of KRN 7000 on Survival Period ofMRL lpr/lpr Mice

Ten animals per group of female MRL lpr/lpr mice were used for thefollowing experiment. MLR mice purchased at the age of 4 weeks wererandomly divided into 2 groups (10 animals per group). A vehicle (aphysiological saline solution containing 0.025%W Polysolvate 20) or KRN7000 (100 μg/kg) was intraperitoneally administered twice a week (onTuesday and Friday) to the animals starting from the age of 5 weeks.Survival of the animals was observed every day.

As shown in FIG. 4, three mice to which KRN 7000 was administeredsurvived even at 350 days after the start of the administration, whileall the mice to which the vehicle was administered died within 250 daysafter the start of the administration.

Pharmacological Test Example 5 Suppression of 4% DSS-Induced MouseColitis by KRN 7000

Ten animals per group of CDF1 mice (6 weeks of age, females) (Japan SLCInc.) were used for the following experiment. Day 0 was defined as theday when a 4% DSS solution (4% (w/v) dextran sodium sulfate (DSS)dissolved in water) was first provided as drinking water. The animalswere divided into 3 groups, i.e., a group to which 100 μg/kg of KRN 7000were intraperitoneally administered on days 1, 5, and 9, a group towhich 1 μg/mouse of IL-12 was intraperitoneally administered on days 1,3, 5, 7 and 9, and an untreated (control) group. Body weight wasmeasured and survival or death of the animals were observed daily.Changes in body weight and survival rate are shown in FIGS. 5A and 5B,respectively.

As shown in FIG. 5A, weight loss was observed at an extremely earlyperiod in the IL-12-administered group as compared with the controlgroup. However, weight loss was obviously observed at a later period inthe KRN 7000-administered group as compared with the control group.

Furthermore, as shown in FIG. 5B, the survival period of theIL-12-treated mice was significantly shorter than that of the controlgroup. However, the survival period of KRN7000-treated mice wassignificantly longer than that of the control group.

4% DSS-induced mouse colitis is a model for human ulcerative colitis(Elson, C. et al., Gastroenterology, 109, 1344 (1995)). Accordingly, theresults mentioned above indicate that KRN 7000 is effective in treatingulcerative colitis.

Pharmacological Test Example 6 Stimulation of NKT Cell Proliferation byCompounds Having α-Glycosylceramide Structure

Stimulation of NKT cell proliferation by compounds having anα-glycosylceramide structure was studied using spleen cells ofRAG-1KO/Vα14tg/Vβ8.2tg mice shown in Pharmacological Test Example 1.

Spleen cells were prepared from the spleen of RAG-1KO/Vα14tg/Vβ8.2tgmice by conventional methods. These spleen cells were suspended at 2×10⁶cells/ml in an RPMI 1640 medium supplemented with 10% FCS, and 100 μleach of the suspension were plated into wells of 96-well round-bottomedplates. Ten different kinds of compounds having an α-glycosylceramidestructure shown in FIG. 12 were added to the wells of the plates at afinal concentration of 1, 10 or 100 ng/ml, and the plates were incubatedfor 2 days. Sixteen hours after the addition of [³H] thymidine (0.5μCi/well), the cells were harvested. The amount of [³H] thymidineincorporated into the cells was measured by a liquid scintillationcounter. Results are shown in Table 2.

TABLE 2 [³H] Thymidine incorporation (cpm) Sample 1 (ng/ml) 10 (ng/ml)100 (ng/ml) Vehicle 2090 2056 2014 KRN7000 40064 74669 102543 AGL5173176 15583 83169 AGL563 2063 3773 13131 AGL571 3969 17848 118092 AGL5772083 7792 49701 AGL586 5137 39750 102425 AGL584 29331 65084 96783S1140B-9 3387 10265 49520 719-7 5287 30179 60528 STL-8 4761 26474 47141

As shown in Table 2, all the compounds above were revealed to have asignificant activity to stimulate NKT cell proliferation at aconcentration of 100 ng/ml as compared with the vehicle-added group.

The results mentioned above indicate that glycosidic compounds having anα-D-glycosylceramide and glycosidic compounds havingα-D-glycosylceramide, in which other sugar is bound to its sugar moiety,are effective in treating autoimmune diseases.

Pharmacological Test Example 7 Suppression of the Onset of ExperimentalAutoimmune Encephalomyelitis by KRN 7000

Ten animals per group of C57BL/6 mice (6-week-old females) were used forthe following experiment. 200 μg of a partial peptide of myelinoligodendrocyte glycoprotein (MOG33-55) and 500 μg of Mycobacteriumtuberculosis H37Ra were added to Freund's incomplete adjuvant to preparean emulsion. Mice were immunized by subcutaneously injecting thisemulsion on day 0 and day 7. Further, 500 ng of pertussis toxin wereintraperitoneally administered on day 1 and day 2 to induce experimentalautoimmune encephalomyelitis (EAE) in mice. The animals were dividedinto 2 groups, i.e., a group to which 20 μg/kg of KRN 7000 wereintraperitoneally administered on days 1, 5, 8, 12 and 15 and a group towhich a vehicle (0.5%, Polysolvate 20) was administered in a similarmanner. The level of EAE symptoms was observed every day. The level ofEAE symptoms of individual mice in each group was shown in FIG. 6.

As shown in FIG. 6, in the vehicle-administered group (FIG. 6A), all themice showed the EAE onset within 15 days after the first MOG peptideimmunization, and 80% of them died. However, in the KRN7000-administered group (FIG. 6B), 4 out of 10 mice showed the EAEonset, and only 2 of them died.

The results mentioned above indicate that KRN 7000 suppressed the EAEonset in mice. The EAE is a model for human multiple sclerosis (MS)(Autoimmune Disease Models, edited by Cohen I. R. and Miller A.,Academic Press, Inc. (1994), Chapter 1, p. 1). Therefore, the resultsmentioned above indicate that KRN 7000 is effective in treating multiplesclerosis.

Pharmacological Test Experiment 8 Suppression of Mouse Diabetes Onset byKRN 7000

Ten animals per group of NOD/ShiJic (NOD) mice (6-week-old, females)(Japan Clea, Inc.) were used for the following experiment. NOD miceshows the onset of diabetes with aging. The animals were divided intotwo groups, i.e., one group to which 100 μg/kg of KRN 7000 wereintraperitoneally administered twice a week starting from 7-weeks ofage, and an untreated control group. The presence and absence ofdiabetic symptoms was examined every week. The blood glucose level wasmeasured using a glucometer (Miles Sankyo), and mice showing the valueof more than 200 mg/dL twice consecutively were diagnosed to bediabetic. FIG. 7 shows the incidence of diabetic mice in the two groups.

As shown in FIG. 7, none of the KRN 7000-administered mice becamediabetic even at the age of 35 weeks whereas 80% of the mice in thecontrol group became diabetic at the age of 35 weeks.

The results mentioned above indicate that KRN 7000 suppresses thespontaneous onset of diabetes in NOD mice. The NOC mouse is a modelanimal for human type I diabetes (Autoimmune Disease Models, edited byChoen I. R. and Miller A., Academic Press, Inc. (1994), Chapter 9, p.149). The results mentioned above indicate that KRN 7000 is effective intreating type I diabetes.

Pharmacological Test Example 9 Stimulation of Vα24⁺ NKT CellProliferation by KRN 7000

Peripheral blood mononuclear cells of a normal human were cultured for 4days in an AIM medium supplemented with 10% FCS with the addition ofGM-CSF (400 U/ml), IL-4 (200 U/ml) and KRN 7000 (100 ng/ml) to prepareantigen-presenting cells.

An autologous mixed leukocyte reaction (MLR) was performed using theseantigen-presenting cells as stimulator cells and autologous peripheralblood mononuclear cells as responder cells. After incubation for 10days, IL-2 (5 U/ml) was added, incubation was continued for another 4days, and the cells were harvested. Next, CD4, CD8 double negative cellswere recovered from these harvested cells and subjected to the phenotypeanalysis. The phenotypic analysis was expressed by fluorescenceintensity of labeled antibodies against cells having various phenotypes.Results are shown in FIG. 8.

As shown in FIG. 8, it was revealed that a large number of cells havingphenotype CD4⁻CD8⁻CD3⁺Vα24⁺Vβ11⁺NKRP1A⁺ (a subset of Vα24⁺ NKT cells)were present in these cell groups.

The cells were cultured using IL-2 (5 U/ml) to stimulate theproliferation of Vα24⁺ NKT cells, which were used as responder cells inthe following autologous mixed leukocyte reaction. Autologous peripheralblood mononuclear cells were cultured for 4 days with the addition ofGM-CSF+IL-4 and 100 ng/ml of KRN 7000, AGL-583 (β-galactosylceramide,β-GalCer) or 0.1% DMSO (vehicle) to prepare antigen-presenting cells. Anautologous mixed leukocyte reaction was carried out using theseantigen-presenting cells as stimulator cells. After incubation for 2days, [³H]thymidine (0.5 μCi/ml) was added, and cells were harvestedafter 8 hours to measure [³H]thymidine uptake into the cells by a liquidscintillation counter. Results are shown in FIG. 9.

As shown in FIG. 9, antigen presenting cells treated with the vehicle orβ-galactosylceramide showed no effect on Vα24⁺ NKT cell proliferationwhile antigen presenting cells treated with KRN 7000 showed a markedstimulative effect on Vα24⁺ NKT cell proliferation in a manner dependenton the number of antigen-presenting cells. Furthermore, inhibitoryeffects of anti-CD1a, CD1b, CD1c, and CD1d antibodies on the stimulationof Vα24⁺ NKT cell proliferation by the KRN 7000-treatedantigen-presenting cells were assessed. As a result, only anti-CD1dantibody inhibited the stimulation of Vα24⁺ NKT cell proliferation (datanot shown).

The results mentioned above indicate that KRN 7000 is effective instimulating the proliferation of human Vα24⁺ NKT cells, a counterpart ofmouse Vα14⁺ NKT cells. Considering the current report that patients withadvanced type I diabetes have an extremely small number of Vα24⁺ NKTcells (Wilson et al., Nature, 391, 177 (1998)) and the results ofPharmacological Test Examples 3, 7 and 8, the results strongly suggestthat KRN 7000 is effective in preventing or treating autoimmunediseases, in which human Vα24⁺ NKT cells are involved, such as systemiclupus erythematosus, systemic sclerosis, multiple sclerosis and type Idiabetes.

Pharmacological Test Example 10 Induction of Abortion by KRN 7000

C57BL/6 mice (Japan SLC) were used for the following experiment. The daywhen a vaginal plug was observed was defined as day 0. Pregnant micewere divided into 2 groups (6 mice per group), i.e., one group to which150 μg/kg of KRN 7000 were intravenously administered on days 7, 8 and9, and another to which PBS (phosphate- buffered saline) wasadministered in the same way. On day 12, the uterus was removed toobserve the state of the embryo.

In the PBS-administered group, of the total of 53 embryos, 3 were deadand resorbed. The abortion rate was therefore 5.6%. which is consistentwith the natural abortion rate of C57BL/6 mice (about 5%).

In the KRN 7000-administered group, of the total of 36 embryos, 12 weredead and resorbed. The abortion rate was 33%, which was obviously higherthan that for the untreated group.

When the same amount of KRN 7000 was administered to Vα14⁺ NKT-deficient(Jα281KO) mice (Kawano, T. et al., Science, 278, 1626-1629 (1997)) inthe same manner, the abortion rate was 5.3%, which was equivalent tothat for the PBS-administered group (5.0%). The Vα14⁺ NKT-deficient miceare available from Masaru Taniguchi, School of Medicine, ChibaUniversity.

Considering that KRN 7000 activates NKT cells, a significant correlationbetween NKT cell activation and abortion induction was indicated.

IL-12 is also known to activate NKT cells. When the same amount of IL-12was administered to mice ((CBA×DBA/2)F₁) in the same manner, theresulting abortion rate was 34%. This result strongly supports thecorrelation mentioned above.

The results mentioned above indicate that KRN 7000 and IL-12 each havean ability to induce abortion.

Pharmacological Test Example 11 Acute Toxicity Test by a SingleAdministration

The compound of Example 1 was intravenously administered to mice.Results showed that LD₅₀ for the compound was more than 10 mg/kg.Furthermore, the compound has a low toxicity showing no particularsymptom at the administration level of 10 mg/kg.

What is claimed is:
 1. A method for treating an autoimmune diseasecomprising the step of administering a compound of formula (I) or a saltor a solvate thereof to a mammal including a human in need thereof:

wherein R¹ to R⁹ and X are as defined wherein R¹ represents H or OH, Xrepresents an integer between 7 and 27, R² represents a substituentselected from the group consisting of the following (a) to (e), whereinY represents an integer between 5 and 17: (a) —CH₂(CH₂)_(y)CH₃ (b)—CH(OH)(CH₂)_(y)CH₃ (c) —CH(OH)(CH₂)_(y)CH(CH₃)₂ (d) —CH═CH(CH₂)_(y)CH₃(e)—CH(OH)(CH₂)_(y)CH(CH₃)CH₂CH₃, and R³ to R⁹ represent substituents asdefined in any one of the following (i) to (ii): (i) when R³, R⁶ and R⁸represent H, R⁴ represents H, OH, NH₂, NHCOCH₃, or a substituentselected from the group consisting of the following groups (A) to (D):

R⁵ represents OH or a substituent selected from the group consisting ofthe following groups (E) and (F):

R⁷ represents OH or a substituent selected from the group consisting ofthe following groups (A) to (D):

R⁹ represents H, CH₃, CH₂OH or a substituent selected from the groupconsisting of the following groups (A) to (D):

(ii) when R³, R⁶ and R⁷ represent H, R⁴ represents H, OH, NH₂, NHCOCH₃,or a substituent selected from the group consisting of the followinggroups (A) to (D):

R⁵ represents OH or a substituent selected from the group consisting ofgroups (E) and (F):

R⁸ represents OH or a substituent selected from the group consisting ofthe following groups (A) to (D):

and R⁹ represents H, CH₃, CH₂OH or a substituent selected from the groupconsisting of the following groups (A) to (D):


2. A method as claimed in claim 1 wherein the autoimmune disease issystemic lupus erythematosus or systemic sclerosis.
 3. A method asclaimed in claim 2 wherein R³ and R⁶ represents H, R⁴ represents OH or asubstituent of any one of groups (A) to (D), R⁵ represents OH or asubstituent of group (F) or (F), R⁷ and R⁸ each represent H or OHwherein both R⁷ and R⁸ do not represent the same substituent, and R⁹represents CH₂OH, CH₃, H or a substituent of any one of groups (A′) to(D′).
 4. A method as claimed in claim 2 wherein X represents an integerbetween 21 and 25 and R² represents substituent (b) wherein Y representsan integer between 11 and
 15. 5. A method as claimed in claim 2 whereinX represents an integer between 9 and 13 and R² represents substituent(a) wherein Y represents an integer between 11 and
 15. 6. A method asclaimed in claim 2 wherein a compound of formula (I) is(2S,3S,4R)-1-(αD-glactopyransoyloxy)-2-hexacosanoylamino-3,4-octadecanediol.7. A method as claimed in claim 1 wherein the autoimmune disease isulcerative colitis.
 8. A method as claimed in claim 7 wherein R³ and R⁶represents H, R⁴ represents OH or a substituent of any one of groups (A)to (D), R⁵ represents OH or a substituent of group (F) or (F), R⁷ and R⁸each represent H or OH wherein both R⁷ and R⁸ do not represent the samesubstituent, and R⁹ represents CH₂OH, CH₃, H or a substituent of any ona of groups (A′) to (D′).
 9. A method as claimed in claim 7 wherein Xrepresents an integer between 21 and 25 and R² represents substituent(b) wherein Y represents en integer between 11 and
 15. 10. A method asclaimed in claim 7 wherein X represents an integer between 9 and 13 andR² represents substituent (a) wherein Y represents an integer between 11and
 15. 11. A method as claimed in claim 7 wherein a compound of formula(I) is(2S,3S,4R)-1-(αD-glactopyransoyloxy)-2-hexacosanoylamino-3,4-octadecanediol.12. A method as claimed in claim 1 wherein the autoimmune disease isencephalomyelitis or multiple sclerosis.
 13. A method as claimed inclaim 12 wherein R³ and R⁶ represents H, R⁴ represents OH or asubstituent of any one of groups (A) to (D), R⁵ represents OH or asubstituent of group (B) or (F), R⁷ and R⁸ each represent H or OHwherein both R⁷ and R⁸ do not represent the same substituent, and R⁹represents CH₂OH, CH₃, H or a substituent of any one of groups (A′) to(D′).
 14. A method as claimed in claim 12 wherein X represents aninteger between 21 and 25 and R² represents substituent (b) wherein Yrepresents an integer between 11 and
 15. 15. A method as claimed inclaim 12 wherein X represents an integer between 9 and 13 and R²represents substituent (a) wherein Y represents an integer between 11and
 15. 16. A method as claimed in claim 12 wherein a compound offormula (I) is(2S,3S,4R)-1-(αD-glactopyransoyloxy)-2-hexacosanoylamino-3,4-octadecanediol.17. A method as claimed in claim 1 wherein the autoimmune disease istype I diabetes.
 18. A method as claimed in claim 17 wherein R³ and R⁶represents H, R⁴ represents OH or a substituent of any one of groups (A)to (D), R⁵ represents OH or a substituent of group (E) or (F), R⁷ and R⁸each represent H or OH wherein both R⁷ and R⁸ do not represent the samesubstituent, and R⁹ represents CH₂OH, CH₃, H or a substituent of any oneof groups (A′) to (D′).
 19. A method as claimed in claim 17 wherein Xrepresents an integer between 21 and 25 and R² represents substituent(b) wherein Y represents an integer between 11 and
 15. 20. A method asclaimed in claim 17 wherein X represents an integer between 9 and 13 andR² represents substituent (a) wherein Y represents an integer between 11and
 15. 21. A method as claimed in claim 17 wherein a compound offormula (I) is(2S,3S,4R)-1-(αD-glactopyransoyloxy)-2-hexacosanoylamino-3,4-octadecanediol.22. A method for inducing abortion which comprises the step ofadministrating a compound of formula (I) or a salt or a solvate thereofto a mammal including a human in need thereof:

wherein R¹ to R⁹ and X are as defined wherein R¹ represents H or OH, Xrepresents an integer between 7 and 27, R² represents a substituentselected from the group consisting of the following (a) to (e), whereinY represents an integer between 5 and 17: (a) —CH₂(CH₂)_(y)CH₃ (b)—CH(OH)(CH₂)_(y)CH₃ (c) —CH(OH)(CH₂)_(y)CH(CH₃)₂ (d) —CH═CH(CH₂)_(y)CH₃(e)—CH(OH)(CH₂)_(y)CH(CH₃)CH₂CH₃, and R³ to R⁹ represent substituents asdefined in any one of the following (i) to (ii): (i) when R³, R⁶ and R⁸represent H, R⁴ represents H, OH, NH₂, NHCOCH₃, or a substituentselected from the group consisting of the following groups (A) to (D):

R⁵ represents OH or a substituent selected from the group consisting ofthe following groups (B) and (F):

R⁷ represents OH or a substituent selected from the group consisting ofthe following groups (A) to (D):

R⁹ represents H, CH₃, CH₂OH or a substituent selected from the groupconsisting of the following groups (A) to (D):

(ii) when R³, R⁶ and R⁷ represent H, R⁴ represents H, OH, NH₂, NHCOCH₃,or a substituent selected from the group consisting of the followinggroups (A) to (D):

R⁵ represents OH or a substituent selected from the group consisting ofgroups (E) and (F):

R⁸ represents OH or a substituent selected from the group consisting ofthe following groups (A) to (D):

and R⁹ represents H, CH₃, CH₂OH or a substituent selected from the groupconsisting of the following groups (A) to (D):


23. A method as claimed in claim 22 wherein R³ and R⁶ represents H, R⁴represents OH or a substituent of any one of groups (A) to (D), R⁵represents OH or a substituent of group (F) or (F), R⁷ and R⁸ eachrepresent H or OH wherein both R⁷ and R⁸ do not represent the samesubstituent, and R⁹ represents CH₂OH, CH₃, H or a substituent of any oneof groups (A′) to (D′).
 24. A method as claimed in claim 22 wherein Xrepresents an integer between 21 and 25 and R² represents substituent(b) wherein Y represents an integer between 11 and
 15. 25. A method asclaimed in claim 22 wherein X represents an integer between 9 and 13 andR² represents substituent (a) wherein Y represents an integer between 11and
 15. 26. A method as claimed in claim 22 wherein a compound offormula (I) is(2S,3S,4R)-1-(αD-glactopyransoyloxy)-2-hexacosanoylamino-3,4-octadecanediol.27. A method as claimed in claim 1 wherein R³ and R⁶ represents H, R⁴represents OH or a substituent of any one of groups (A) to (D), R⁵represents OH or a substituent of group (E) or (F), R⁷ and R⁸ eachrepresent H or OH wherein both R⁷ and R⁸ do not represent the samesubstituent, and R⁹ represents CH₂OH, CH₃, H or a substituent of any oneof groups (A′) to (D′).
 28. A method as claimed in claim 1 wherein Xrepresents an integer between 21 and 25 and R² represents substituent(b) wherein Y represents an integer between 11 and
 15. 29. A method asclaimed in claim 1 wherein X represents an integer between 9 and 13 andR² represents substituent (a) wherein Y represents an integer between 11and
 15. 30. A method as claimed in claim 1 wherein a compound of formula(I) is(2S,3S,4R)-1-(α-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecanediol.